Despite the importance of the thymic environment for the generation of T cells and the projection of self that helps define self-tolerance for developing T cells, much of the differentiation program that gives rise to thymic epithelium remains poorly understood. An important advance in this area has been the appreciation that this epithelium is mitotically active and that it is continually replaced postnatally. This means that the composition of thymic epithelium represents a mixture of epithelial cells at different stages of differentiation. Presently, the means to parse this epithelial heterogeneity is inadequate and this has hampered efforts to define the program of epithelial differentiation and to relate this process to the expression of Aire, which influences the ability of thymic epithelial cells to present tolerogenic self antigens. Our previous work has demonstrated that the MTEC compartment of the Aire-/- thymus exhibits features consistent with an altered program of MTEC differentiation, with expansion of a MTEC subset that is rare in the WT thymus. We propose here to generate reagent antibodies against enriched populations of MTEC from Aire+/+ and Aire-/- thymi as a means to develop reagent antibodies that will allow us to define MTEC heterogeneity with more precision. These new tools will support our long-standing efforts to establish precursor-progeny relationships within the thymic epithelial compartment and will help efforts to understand how different mutations that affect thymic epithelial organization are affecting thymic epithelial differentiation. PUBLIC HEALTH RELEVANCE: Epithelial cells comprising the thymic environment play a critical role in the generation and differentiation of T cells. Lack of knowledge regarding the basis for the observed heterogeneity of thymic epithelium is impeding the development of strategies to retard age-related loss of thymic function or to enhance thymic expression of molecules that could modulate the ability to establish self-tolerance. This project will generate much needed antibody tools to help define thymic epithelial heterogeneity and relate the expression of these molecules to several models of perturbed thymic epithelial differentiation.